The partial purification of the lecithinase of Clostridium Hemolyticum
by Donald Edward McRoberts
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Chemistry
Montana State University
© Copyright by Donald Edward McRoberts (1962)
Abstract:
The possibility of isolating the lecithinase of Clostridium hemoivticum from other proteins in the
culture filtrate by differences in the solubility of the various components in acid solutions was
investigated.
The lecithinolytic activity of the enzyme per milligram of nitrogen was used as a measure of its purity.
Variables such as polymetaphosphate concentration, acidity, temperature, agitation, and acid contact
time, which were thought likely to influence the amount of the precipitate, were investigated. The
stability of the enzyme alone and in the presence of polymetaphosphate at certain pH values has been
estimated in a number of chemical systems under various physical conditions.
The amount of the enzyme which can be recovered by acid precipitation from a solution with and
without polymetaphosphate at various concentrations, pH values, and temperature has been evaluated.
An attempt has also been made to purify the crude lecithinase by separation on the
N,N-Diethylaminoethylcellulose column. The amount of crude lecithinase adsorbed on a certain weight
of DEAE cellulose was determined. TRIS (hydroxymethylamino) methane buffer was selected for ruse
on the column. Calcium ion was found to stabilize the lecithinase during dialysis of the eluate.
Purification values for acid precipitation of the lecithinase -PO3 and DEAE cellulose column
separation were determined. Acid purification of the crude lecithinase was typically about 22 fold for
crude lecithinase which had been, purified about 10 fold in previous treatments; i.e., a total of 220 fold.
Separation of the DEAE cellulose column gave about five fold purification. This value is estimated,
since several lecithinase peaks were obtained on the graph when lecithinase activity was plotted against
eluate tube number. It is thought that these several peaks may indicate that Clostridium hemoivticum
elaborates more than one lecithinase.
THE PARTIAL PURIFICATION OF THE LECITHINASE OF
CLOSTRIDIUM HEMOLYTICUM'
by
Donald MoRoberts
A THESIS
Submitted to the Graduate Faculty
in
partial fulfillment of the requirements
for the degree of
Master of Science in Chemistry
at
Montana State College
Approved:
Chairman, Examining^bmmittee
MONTANA STATE COLLEGE
Bozeman, Montana
December, 1962
Tlii.ACKNOWffiDGEMEliJT
I wish to express ray appreciation to Dr. K. F. Swingle and Dr. R. F .
Keeler for assistance and advice in collecting the data and preparing the
thesis in its final form,, to Dr. L„ D. S. Smith and Mr. K. D. Claus9 at the
Montana Veterinary Research Laboratory9 for assistance in growing the
Clostridium hemolvticum bacteria.
I wish to thank the members of the
Chemistry Research Department at Montana State College for the use of equip­
ment and chemicals.
The .work performed in the development of this thesis was done in
partial fulfillment of Contract.N6onrT2'379 Task Order Il9 Project NR 134 244
between the Office of Naval Research and Montana State College.
-iv-
TABLE OF CONTENTS
SUBJECT
PAGE .NUMBER. .
Introduction
1
Historical
Acid precipitation of bacterial toxins.
Polymetaphosphate and protein.
N,N Diethylaminoethyl cellulose column.
Materials and methods
General analytical methods.
Preparation of materials
Analysis of crude lecithinase
-Part I:
( A.
Acid precipitation of the lecithinase-PO^ complex as
a means of increasing the specific activity of the
lecithinase.
EXPERIMENTAL PROCEDURES
I, 'Stability of lecithinase in the presence of
polymetaphosphate in acid solutions.
2„ -Effect of pH on the stability of lecithinasePO3 .
3. Lecithinase purification as a function of acid contact time.
4. -The pH at which'the lecithinase-P03 precip­
itate forms for one ratio of PMP and protein.
5. .Purification of lecithinase in,.bacterial
filtrate at acid pH values.
6. Concentration of components in the crude lecir
thinase-POg precipitate as a function of con­
centration of polymetaphosphate and the pH of
the solution.
B. RESULTS
C.
DISCUSSION
1. Protective action of PMP on!.lecithinase.
2. The stability of Iecithinase-PO3 in 2-methyl2-amino-l,3 propanediol-acetic acid buffer.
3. ■Lecithinase purification as a function of
acid contact time.
4. The separation of lecithinase protein from
nonlecithinase protein.
5. ,Purification of lecithinase by reprecipitation.
2
3
4
6
10
16
17
17
18
19
19
20
20
22
40
41
42
42,
43
-V—
6.
7.
8.
9.
10.
D.
CONCLUSIONS
Part II:
A.
B.
C.
D.
Influence of PMP concentration oh the
composition of the acid precipitates.
Influence of pH on the amounts of various
components in the precipitate. 1
'
Interpretation of data in Tables, I and II as
plotted in Figures 8 to 14.
The MLD per mg. of phosphorus ratio as in­
fluenced by the amount of PMP.
Nitrogen and phosphorus in the precipitate
as a function of solution pH.
The separation of crude lecithinase on the N 3N diethyIaminoethyIcellulose column.
43
44
44
45
46
48
49
EXPERIMENTAL PROCEDURE
1. -The column material.
2. Eluent buffers.
3. .Protein placed on the column.
4+ Connecting tubing.
5. Mixing chamber.
6. Column and. dialysis operation.
49
49
50
52
52
52
RESULTS
54
„
DISCUSSION
CONCLUSIONS
61
64
General Conclusions
64
Literature Cited
66
=-vi=rLIST OF TABLES
TABLE
I
II
ITI
IV
V
PAGE
NUMBER
The concentration of Iecithinase9 nitrogen9 and
phosphorus in precipitates obtained at three pH
values arid various concentrations of polymeta­
phosphate at room temperatures and atS-Ey0C 0
25
Analysis of variance of the data from Table T
Summary of Fdistributionsignificance,
28
The composition of eluent buffers for the diethylaminoethyl cellulose column.
'
50
The purification of crude lecithinase previous
to placing it on the diethylaminoethyl cellu­
lose column.
51
.-Analysis of column eluates for lecithinase and
protein,
55
-vii-
LIST OF FIGURES
FIGURE
'
PAGE
NUMBER_______ SUBJECT ______________ _______________________________ NUMBER
1,2,3
4
5
6
7
8
9
10
11
12
13
Lecithinase recovery in solutions at acid pH
Values with and without PMP0
29
Enzyme recovery in 2-methyl-2-amino-I ,3 pro­
panediol-acetic acid buffer at two acid con­
tact times and two temperatures,
30
Lecithinase purification in the sodium polyl­
inetaphosphate precipitate as a function of
acid contact time at 3-5°C,
31
Lecithinase and nitrogen recovery in precipitates.
32
■ Recovery of lecithinase by repeated precipitation
in a glycerol solution at pH 2.8.
. 32
Lecithinase activity per mg. of nitrogen as,a
function of concentration of SPMP at constant
pH and room temperature.
33
.Lecithinase activity per mg. of nitrogen as a
function of concentration of SPMP at constant
.pH and 3-5°C.‘
'
34
Lecithinase activity per mg. of phosphorus as a
function of the SPMP concentration at a const­
ant pH and room temperature.
35
Lecithinase activity per mg. of phosphorus as a
function of concentration of SPMP at constant
,pH and 3-5°C.
'
36
Protein nitrogen in the precipitate a-s a function
of solution pH in the presence of 2 meq. of poly­
metaphosphate per g. of protein.
37
Amount of phosphorus in the precipitate as a
function of pH in the presence of 2 meq. of
PMP per g. of protein.
38
-viii14
15
•P/K( ratio as a function of SPMP concentration
at constant pH and room temperature.
39
Hydrogen ion concentration as pH in the eluate
fractions.
57
16
Ionic strength of column eluates.
17
Analysis of column eluates for leclthinase
activity.
59
Xowry protein concentration in column eluates.
60
18
' 58
-Ix-
ABSTRACT
The possibility of isolating the lecithinase of Clostridium
hemoivticum from, other proteins in the culture filtrate by differences
in the solubility of the various components in acid solutions was
investigated.
The lecithinolytic activity of the enzyme per milligram of nitrogen
was used as a measure of its purity. Variables such as polymetaphosphate
concentration, acidity, temperature, agitation, and acid contact time,
which were thought likely to influence the amount of the precipitate, were
investigated.
The stability of the enzyme alone and in the presence of poly­
metaphosphate at certain pH values has been estimated in a number of chemical
systems under various physical conditions.
The amount of the enzyme which can be recovered by acid precipitation
from a solution with and without polymetaphosphate at various concentrations,
pH values, and temperature has been evaluated.
An attempt has also been made to purify the crude lecithinase by
separation on the N',N-Diethylaminoethylcellulose column. The amount of
crude lecithinase adsorbed on a certain weight of DEAE cellulose was.
determined.
TRIS (hydro xyme thyIamino) methane buffer was selected for ruse
on the column. Calcium ion was found to stabilize the lecithinase during
dialysis of the eluate.
Purification values for acid precipitation of the lecithinase -POg ,
and DEAE cellulose column separation were determined. Acid purification
of the crude lecithinase was typically about 22 fold for crude lecithinase
which had been, purified about 10 fold in previous treatments; i.e., a
total of 220 fold. Separation of the DEAE cellulose column gave about five
fold purification. This value is estimated, since several lecithinase
peaks were obtained on the graph when lecithinase activity was plotted
against eluate tube number. It is thought that these several peaks may
indicate that Clostridium hemoivticum elaborates more than one lecithinase.
INTRODUCTION
Immunization of cattle against bacillary icterohemoglobinurias. or
"red water"' disease, for periods longer than six months has not been
achieved.
This disease is caused by the bacterium Clostridium hemdlvticum
(i, 2) which produces a lethal hemolytic toxin, shown by Jasmin (.3) to
be a lecithinase.
S1Wingle (4) has shown that the enzyme has lecithinolytic
activity since it produce's neutral fat and phosphoryl choline.
The action
of the enzyme is similar, then, to the Lecithinase A elaborated by
Clostridium perfrinaens (Mac Farlane and Knight)(5).
As a first step towards developing an improved immunizing agent,,
an attempt was made to isolate this lecithinase in greater purity than
had hitherto been achieved.
Previous attempts (6) at this isolation, using ammonium sulfate frac­
tionation, nucleic acid precipitation, or adsorption on resin such as
IRC-50 or on magnesium oxide, failed to yield a product of desired purity.
In this study, two additional methods for isolation of the lecithinase were
investigated:
acid precipitation of the lecithinase in the presence of a
protective agent,, and the separation of crude lecithinase on an N,Ndiethylaminoethylcellulose column.
Acid Precipitation of Lecithinase;
Turpin, Raynaud and Relyveld (7) were able to concentrate effectively
the toxins and toxoids of tetanus, diphtheria-, and staphylococci, and the
toxins of Clostridium perfrinaens.'Clostridium sordellii. and Clostridium
botulinum by acid precipitation in the presence of spdium polymetaphosphate
or ethylenediaminetetraacetic acid.
Their studies did not include a con­
sideration of Clostridium hemolvticum lecithinase.
Since Swingle (4) has
-2-
shown that the lecithinase of Clostridium hemolvticum is rapidly inactivated
by acid, the possibility of its being protected by an agent such as. sodium
polymetaphosphate before acid precipitation was investigated.
Even though
such protection was not in general achieved, a number of observations were
made regarding the stability of the enzyme under various conditions, its. '
reactions with sodium polymetaphosphate, and its precipitation by acids.
The first separation of crude lecithinase on the diethylaminoethylcellulose column showed that lecithinase activity occurred in more than one
series of eluate tubes.
When a profile curye was plotted showing lecithinase
activity against eluate tube number, several peaks of lecithinase activity
appeared on the curve.
It was. then assumed that these peaks represented the
action of different species of lecithinase- protein.
As such, an effort was
then made to demonstrate that two or more. Iecithinases were elaborated by
Clostridium hemolvticum bacteria.
No further effort was made to isolate
the lecithinase from the crude lecithinase mixture by the procedure of acid
precipitation.
HISTORICAL
Acid Precipitation of Bacterial Toxins.?
Much research has been done on the possibility of concentrating
toxins or toxoids by acid precipitation.
Schmidt, Hansen, and Kjger (8)
in 1931 used hydrochloric acid on diphtheria toxin and toxoid, but ob­
served a destruction of the active principle of the toxin or toxoid.
Burnet and Freeman (9) purified the toxin of staphylococci by precipitation
with acetic acid.,
Nelis (10) ,et al_. studied the action of strong acids on
-3-
- staphylococcal toxin.
With HgSO^, the authors observed partial destruction
of the toxin at room temperature.
Jacobs and Behan (ll) concentrated the
antitoxin of tetanus with trichloroacetic acid and eliminated 99 per cent
of the impurities. .In 1951 Jacobs and Gillis (12) used metaphosphoric acid
for the concentration of diphteria toxoid to eliminate 97 per cent of the
protein nitrogen.
Turpin, Raynaud and Relyveld (7) improved the yield of toxin or toxoid
separated by acid precipitation by adding a protective agent.
A partial
history of one of their protective agents,, sodium polymetaphosphate,, is
given in the next section.
Polvmetapho sphate and Proteint
M.etaphosphoric acid of soda (13), so. named in the early literature, has
been recognized (13) since 1833 as an agent which will precipitate proteins
(13).
Because of the efficacious action of polymetaphosphoric acid as. a
piotein precipitant, many workers, have attempted to determine the specific
action by which the protein in the presence, of an acid solution of the poly^
mer becomes insoluble.
According to Briggs, (14) Schofield, (15) Perlman, (16, 17) Herrmann
and Perlmann, (18, 19) and Ebel and Colas,. (20) a reaction occurs between
polymetaphosphate and the nitrogen of a nitrogenous compound or protein.
.Briggs (14) has shown by dialysis that at neutral pH values the polymeta­
phosphate can be dialyzed from horse serum albumin, but at acid pH values,
this is not possible.
pho sphoric acid.
Perlmann (17) precipitated egg albumin with meta­
The complex formed crystals which was taken as evidence
that denaturetion did not occur.
-4-
Briggs, (14) using horse serum, albumin in the presence of polymetaphosphate, stated that at low pH values, the ionization of the carboxyl
group is repressed, and the positive charge on the amino group is. com­
pletely neutralised.
The overall effect is a shift of the isoelectric
point in the acid direction.
I
Putnam and Neurath9 (21) working with egg albumin and sodium dodecylsulfate,, stated that precipitation may result from a combination of
oppositely charged electrostatic forces; that is,- an anion detergent will
precipitate protein in the cationic form, but precipitation ceases some­
what above the isoelectric point, even though combination between deter­
gent and protein (22) does occur.
Just above the isoelectric point, (21)
the protein-detergent complex precipitates completely and remains in the
solid state for immediately lower pH values.
The amount of protein precipi­
tating in the presence of detergent depends on (first), the protein:
detergent concentration ratio,
(second) the pH of the solution, (third)
the temperature, and ,(fourth), the ionic strength of the solution.
NfN Diethvlaminoethvl Cellulose Column Chromatbgraohv:
Sober and Peterson (23, 24) first prepared this special cellulose ion
exchanger by treating strongly alkaline cellulose with,2-chlorotriethylamine.
They then used the product in a column for separation of serum
proteins^
Column material of this derivative is said by the authors (23,
24) to have the following properties:
good mechanical strength,, insolu­
bility in water, and, as an anion- exchanger., the ability to adsorb protein
effectively at pH values above the isoelectric point of most proteins.
Hence, elution of the bound protein can be accomplished by increasing the
_ 5,-
ionic strength of the medium or decreasing the pH of the medium or both.
According to Guthrie and Bullock (25) DEAE cellulose^=/ has, a maximum
capacity for protein of 0.7 meq. per g.? and the pH at half capacity is
7.7.
Boman and Westlund'(26) believe that with an anion exchange column,
best results in separation of protein will occur with a cation buffer such
as TRIS (see also ref. 27).
These authors state that DEAE cellulose is
the preferred cellulose derivative for the separation of proteins,, en­
zymes, and some hormones.
It should be mentioned that one disadvantage of using DEAE for sep­
aration of the lecithinase mixture rests in the fact that the material is
an anion exchanger. -On addition of protein to the column the pH of the
eluate increases.
If the protein is. alkali labile, this procedure can be
used only with caution.
Very few lecithinases have been isolated on a DEAE cellulose column.
Of importance to this investigation is the work of Boman and Kaletta (27)
who used DEAE cellulose in TRIS (hydroxymethylamino) methane buffer to
separate three different forms- of phosphodiesterases from phospholipase A
amino acid oxidase.
Sober and Peterson (23) in 1954 reported the separation of two distinct
amidase activities from a lyophilized kidney fraction which differed in their
relative rates toward leucine and alanine amides.
'I/
Diethylaminoethyl cellulose
MATERIALS AND METHODS
General Analytical Methods
Determination of Lecithinase Activity
The determination of the lecithinolytic activity of Clostridium
hemol-vticum was performed according to the method of T u r n e r S w i n g l e and
Strickfaden (28) as follows;
Reagents;
Borate Buffer 2 j
Sodium chloride
Anhydrous calcium chloride
Boric acid
Water to
5.3 g„
2.2 g.
6.2 g.
I liter
The pH was adjusted .to 7*4 with 40% sodium hydroxide.
Standard Solution of Lecithinase in Glvcerol;
A quantity of the culturel filtrate (prepared as. described on^lO-12)
was dialyzed in cellophane tubing at room temperature against three daily
changes of glycerol.
The glycerol solution of the protein was then stand-,
aidized by injecting graded amounts, int,o the lateral tail veins of 35 g.
mice and noting death or survival within 24 hours.
Ifg for instance^ 0.5 ml.
of toxin diluted 1:50 killed all mice but 0.4 ml. of the same toxin did not
kill any$ then the original toxin solution was labeled 100 mouse minimum
lethal dosage (MLD) per ml.
The lecithinase solution which was used in
column work was not standardized by mouse injection.
2/ Also referred to as isotonic borate buffer
* ■ ’~l~or‘
Egg. Yolk Substrate;
One egg yolk was uniformly suspended in 2.5 I.. of borate buffer
which was preserved with three or four large crystals of thymol or I part
Roccal
3/
to 375,000 parts of buffer.
Roccal did not protect the substrate
from decomposition for as long as. thymol.
The mixture was allowed to stand
for one day at room temperature, centrifuged., filtered through a mat of
Super-Cel ^
diatomaceous earth* and allowed to stand for another day.
The
suspension was then refiltered until the turbidancy of the solution as read
against a water blank falls between 0.04 and 0.15 at 650 my.as read with a
round tube of 1.9 cm. diameter in a Coleman spectrophotometer.
The sub­
strate was then standardized against the standard solution of toxin in
glycerol by the procedure described below.
Procedure.:
\
A quantity of the enzyme solution in glycerol .was added to borate
buffer^ and dilute base was added if necessary to bring the pH to 7.4.
solution was then diluted to 5.00 ml. with isotonic borate buffer..
The
Five ml.
of egg.yolk substrate was then added and the mixture incubated at 37°C . for
exactly 20 minutes.
The turbidity which developed in the suspension was
read in a spectrophotometer at 650 my against a blank containing 5.0 ml. of
isotonic borate buffer and 5.0 ml. of egg yolk substrate.
An almost linear
relationship existed between thg quantity of lecithinase and the turbidity
developed.
3/
Roccal - Registered trade mark, Benzalkonium chloride (alkyldimethylammoniumchloride).
^
4/ Super-Cel5 Registered trade mark.
™8-
The activity of the unknown was converted to MLD from the standard
curve which was plotted daily for the particular batch of substrate used in •
the assay.
The variance between successive identical lecithinase assays was
found to be 0.02 MLD„
Calcium ion, which is essential for the action of the lecithinase
on its substrate, is not only precipitated by the polymetaphosphate (34),
but the precipitate itself contributes to the measured turbidity of the
system.
When the amount of PMP introduced into the.assay tube was, less than
0.1 mg., only a small error resulted in the lecithinase analysis.
Swingle (4)
found that maximum lecithinase activity occurs in a calcium ion range between
0.0Q6 M to 0.04 M.
In precipitates of relatively-high specific activity to
phosphate ratios, the polymetapho$phate interference was negligible.
Determination of Nitrogen or Protein
Acid Precipitation Studies:
Nitrogen was determined by the manometric method of Van Slyke (29).
The only modification employed was that, after digestion and boiling down
to the first appearance of white fumes, 5 ml. of water was added to insure
the decomposition of the potassium peroxydisulfate.
Crude Lecithinase Placed on the Diethvlaminoethvlcellulose Columns
Crude lecithinase was first analyzed for protein content by the
Biuret method (30).
The Biuret color intensities were read on the Coleman
or Beckman spectrophotometer at 540 mp.
A curve, Curve 1 ?. was then plotted
of absprbance vs. milliliters of protein solution.,
-9-
According to Gornallil (30) I mg. of protein dissolved in 3 ml. of
water in a I Cjmd cell will give an absorbance of 0.151.
From Curve Is the
yolume of protein solution giving an absorbance of 0,151 was determined^
and the concentration of. the solution in mg. of protein per ml. calculated.
Then appropriate volumes of the protein solution were treated with the
Lowry reagents, (31) and the absorbance obtained was used to plot Curve 2
(absorbance vs. ml. of protein solution).
protein
From Curve 2, the volumes of
solution required for qn absorbance of 0.1, 0.2, 0.3, and 0.4
were read.
Then the weight of protein in these solution volumes was cal­
culated from the Biuret value of mg. of protein per ml. and a new curve,
Curve 3, was plotted using the absorbance values of Curve 2 against mg.
or yg. of protein contained in these volumes of solution.
This standard
curve was then used to convert absorbance of a sample of eluate protein
solution from the column, as determined by the Lowry method,, (31) to the
weight of protein which the solution contained.
Diethvlaminoethvlcellulose Column Eluates:
Protein content in column eluates was determined by the method of
Warburg and Christian,- (32) and Loyvry, (31).
In the Warburg method
(32) absorbance of the protein solution at 260 mp and 280 mp was con­
verted to mg. of protein by the use of a monograph.
Miscellaneous Analyses
Nitrogen in DEAE Cellulose and some prbtein determinations, wa6
determined by standard Kjeldahl (33) procedures.
Sodium polymetaphosphate
-10-
was precipitated a,s barium polymetaphosphate and hydrolyzed to ortho­
phosphate according to the method of Jones (34)„
The phosphate was then
determined by the modified method of Fiske and Subbarow .(35)„
Organic
phosphate in the crude lecithinase was hydrolyzed with hydrogen peroxide
or perchloric acid and determined by the modified method of Fiske and
Subbarqw„
Crude Lecithinase:
Culture! filtrate protein, separated by the ammonium sulfate precipi­
tation^ was analyzed for nitrogen and phosphorus according to the methods
already mentioned (29, 33, 35).
Protein-bound hexose was determined by the
method of Lustig and Langer, (36) and Weimer and Moshin (37)„
The presence
of nucleic acids in the protein solution was indicated after consideration
of values obtained in the Warburg
paper chromatography„
ultraviolet absorption procedure and by
The latter procedure involved developing the spot on
paper with Pabst solvents (38) end then viewing the chromatogram with a SL
Mineral light, Model V-41, which provides light at 253.7 mp.
The presence of lipoprotein in crude lecithinase was shown by treat­
ing' a-paper chromatogram with Sudan black (39).
PREPARATION OF MATERIALS
Crude Lecithinase
Culture Medium and Toxin Production:
The crude lecithinase which was used for acid precipitation of leci­
thinase was, prepared from the culturel filtrate of Clostridium hemolvticum
(Montana Veterinary Research Laboratory, strain number 4473).
The bacteria
were grown in the peptic digest medium of Jasmin (3) modified by Swingle
and Smith (40).
-11-
The peptic digest medium (4) was prepared by placing 500 g. of ground
lean beef, 500 g. of beef liver, and 75Q-1000 g. of ground fresh pig
stomach in 2000 ml. of water.
The pH of the mixture was brought to 2
with hydrochloric acid and 10-20 g. of pepsin added.
The mixture was
digested at 50°C. for 12 hours.,, heated on the steam bath at 80-90°C, to
stop peptic action, refrigerated overnight*, and filtered through a mat
consisting of I cm. of Super-Cel (diatomaceous earth).
The pH of the
filtrate was adjusted to about 7.3 with 5 ,N sodium hydroxide and 0.0375
per cent calcium chloride (W per V) was added, the solution heated at 90°C.
for 30 minutes, and the precipitate filtered off through a mat of Super--Cel.
The filtrate was the peptic digest, abbreviated PD.
Two volumes of PD were diluted with I volume of water.
Then proteose
peptone, I per cent,,, and glucose, 0.5 per cent, were added and the solu-
\
tion brought to a pH of 7.6 with sodium hydroxide.
The medium was then
sterilized in an autoclave.
An inoculum of Clostridium hemolvticum was transferred from the stock
culture in Hall's medium £ / into a tube containing about 20 ml. of the com­
plete PD medium.
This was incubated for about 6 hours at 37°C. and poured
into a small flask containing about 200 ml. of fresh medium.
After in­
cubating for the same time interval, this culture was poured into a large
flask three-fourths filled with medium,; which had been recently heated and
then copied to 37°C.
After a growth time of aboyt 6 hours at 37°C., the p'H
of the medium was adjusted to 7.6 with 5 N sodium hydroxide and the liquid
5/ 'H a l l ’s medium is probably similar to Holman's cooked meat medium with
modification.
I
T12“
cooled to about 4°C0
The refrigerated culture was centrifuged at an RCF
of about 2000 until clear, and the supernatant filtered through a mat of
Super-Cel0
The filtrate is referred to as culturel filtrate.
The crude lecithinase used in the more recent work with the DEAE
cellulose column was prepared according to the modified procedure of
Claus (42),
This medium -was prepared as follows:
equal weight of finely
ground liver and water were mixed in a Waring blender.
chloric acid was added to a pH of 2-2,5,
Concentrated hydro­
Then 0,50 g, of Difco pepsin per
100 g» of liver was stirred in the suspension, and the mixture placed in a
water bath at 50°C. for 15 hours*
After cooling the suspension to 15°C„,
it was centrifuged at moderate speed and the supernatant filtered through
a mat, I cm. thick, of Super-Cel,
The pH of the filtrate was then adjusted
to 7,3 with 5 N sodium hydroxide and calcium chloride dihydrate added to a
concentration of O iS mg. per ml. of solution.
.
The solution was steamed to
9d°C, for 30 minutes, cooled, and filtered through Super-Cel.
An equal
volume of water was added,, plus trypticase to I per cent and soluble starch
to 0.5 per cent.
The pH of the solution readjusted to 7.3 with 5 N sodium
hydroxide, after which the medium was autoclaved.
;
After growth of the bacteria and preparation of the culturel filtrate,
the liquid,- if passed through a Seitz filter*, is referred to as the Seitz
filtrate.
6/
Relative centrifugal force (a I pound, mass spun at an RCF of 1500 would
,weigh 1,500 pounds.)
)
13-
Precipitation and Dialysis of Toxins
The crude lecithinase was precipitated from the cultural filtrate
with ammonium sulfate.
The filtrate was first made to 16 per cent by
weight with ammonium sulfate^ and solids, if any, were removed by centri­
fugation, RCF of 1000 and discarded.
Additional ammonium sulfate was then
added to bring the solution to the final content of 31 per cent by weight.
The precipitated lecithinase was collected by centrifugation and dialyzed :
in a Vistex -Z/ membrane against running distilled water at IO0C 1
In some
cases tap water at 3-&°C. was used for 3-4 days until a chemical test for
the ammonium ion or a conductivity test showed the near absence of ammonium
sulfate in the dialysate.
At this stage the nucleic acid separation of Van Heyninger and
Bidwell (43) was used for the protein ,which was to be placed on the DEAE
cellulose column.
The procedure for the Van Heyninger and Bidwell (43)
separation was as follqws:
the dialysee in the Vistex membrane was centri­
fuged for 10 minutes at an RCF equals 1500 after which the precipitate was
discarded.
The pH of the supernatant was adjusted to 7 with 5 N sodium
hydroxide and the solution cooled to near 0°C.
Ice cold I per cent ribose
nucleic acid? in the amount of 4.7 ml. per 100 ml. of lecithinase solution,
was added with stirring, and the pH of the solution adjusted to 4.5 with
6 N acetic acid.
temperature.
The solution was allowed to stand for I hour at this
Centrifuge cups were then removed from the deep freeze,, and
the precipitate was quickly centrifuged from the acetic acid solution at
an RCF of 1500.
Only a small precipitate was. obtained.
7/ Vistex,Registered trade mark.
This precipitate
— 14—
was saved, and the supernatant was readjusted to pH 7 with sodium hydroxide.
Additional I per cent ribose nucleic acid solution was added in the amount
of 14.1 ml. per 100 ml. of lecithinase solution.
The pH of the solution
was then adjusted to 4.5 with 6 N acetic acid, and the suspension allowed
to stand for 1.5 hours at 3-5°C. after which a heavy percipitate was. obtained
The precipitate was separated from the liquid by centrifugation at RCF
equals 1500 and the two precipitates combined in about 100 ml. of ice
cold water which contained 2 drops of 5 N sodium hydroxide to dissolve
the precipitate.
To remove the protamine nucleate, the pH of the solution
was adjusted to neutrality with dilute hydrochloric acid, and ice cold I
per cent protamine sulfate added drop by drop until no further precipitate
of protamine nucleate formed.
After removing the precipitate by .centri­
fugation at RCF equals 1500, I per cent ribpse nucleic acid was. added to
just precipitate any excess, protamine sulfate.
The solution was then
recentrifuged at an RCF equals 1500.
The final solution was light straw-colored, faintly opalescent and
kept frozen at -IO0C. until used.
Almost one year passed before the
protein separation was made on the DEAE cellulose column.
In the acid precipitation part of this investigation, the protein
solution was lyophilized after dialysis,, without the nucleic acid puri­
fication step.
Sodium Polvmetaphosphate;
The polymer was either prepared by the fusion method (34) of Jones
-15-
or purchased as "Calgon'1^
(The analysis is described on page 9 and 10).
N-Die-thvlaminoethvlcellulose:
The Eastman compound number 7392 was used.
This DEAE cellulose was
found to be somewhat hygroscopic and in purified form contained about 0.1'
per cent less nitrogen than Sober)s product (23).
If proteins in the column eluate are to be determined by using the
Lowry method, any decomposition of DEAE cellulose must be avoided since
the Lowry reagents will develop color with N,N-diethylamine.
Moore and
.Lee (45) found that Eastman DEAE cellulose would yield nitrogenous products
to the column eluate.
The DEAE cellulose used.in this investigation was
purified according to their procedure, which involves treating the DEAE
cellulose with I N sodium hydroxide, washing with water to a supernatant
pH of 7, then washing with ethanol and finally with ether.
Buffers:
THIS (hydroxymethylamino) methane (2-amino-;2-hydroxylmethyl-l,3propanediol) buffer (46) has been used on the DEAE cellulose column. (27).
The product used in this investigation was labeled Sigma 7’t 9.
compound was twice crystalized from water.
The
The final product absorbed
in the. ultraviolet in agreement with the value obtained by Boman and
Westlund (26).
(0.05-0.06 for a 0.5 M solution).
/
8/
The commercial preparation, Calgon, is a glass with a ratio of
NagQsPgOs of 1.1:1.
It is made by fusing food grade phosphoric acid
and commercial soda ash and is commonly called hexametaphosphate. The
unadjusted form used in this study does not contain sodium carbonate.
I
-1 6 -
Weights of constituents in 0.1 M buffer as used in acid precipitation
of Iecithinase-POg are:
2-amino-2-methyl-l?3 propanediol
Glacial acetic acid
Distilled water to
10.5 g.
3.0 g.
1.0 I.
ANABASIS OF CRUDE LECITHINASE
A one ml. sample of crude lecithinase as prepared by ammonium sul­
fate precipitation, dialysis of the precipitate, and centrifuging off any
insoluble
tion.
material
was found to contain12*4 mg. ofsolids per ml.of solu­
Of thisweight, 1.07 mg. was protein-bound hexose.
The Biuret
protein value calculated according to Gornall (30) was 2.2 mg.
The calcu­
lated protein value using a factor of 6.25 for the Kjeldahl nitrogen was.
3.8 mg. of protein.
Very likely other nitrogen-containing compounds are
present.
Lipoproteins were shown by a qualitative test involving Sudan lglack
(39).
Electrophoresis of the crude lecithinase at a pH near 8 in the Perkin
Elmer apparatus showed a large number of different proteins, so many
proteins, in fact, that a photograph of the pattern showed broad heavy
bands which gave no useful information.
Light absorption by the Seitz filtrate ^ a t 260 my was intense
indicating the presence of rather large quantities of nucleic acids.
Paper chromatograms of the protein viewed under ultraviolet light further
indicated the presence of nucleic acids.
2 / Medium after growth of Clostridium hemolvticum centrifuged
and run
through diatomaceous earth twice, then through a Seitz filter.
PART Ti
ACID PRECIPITATION OE THE LEOITHINASfe -tPC3 COMPLEX AS MEANS OF
INCREASING THE SPECIFIC ACTIVITY OF THE LECITHINASE
Since one intent of this investigation was to obtain lecithinase with
a high specific activity
it was necessary to determine, optimum physical
or chemical conditions for retention of lecithinase activity in lecithinasePO3 -^^/systems.
This determination of the probable maximum specific activity
of lecithinase as obtained in the precipitate with optinum conditions
requires at least as empirical consideration of the nitrogen, lecithi­
nase and PMP
equilibria existing between soluble and insoluble com­
ponents in the various, systems at certain acid contact times..
Swingle (4) has determined the stability of the impure enzyme in
various systems under certain types of stresses, such as temperature,
agitation, and adverse hydrogen ion concentration.
The lecithinaSe-PO3
complex was, not investigated.
A.
I.
EXPERIMENTAL PROCEDURES
Stability of the Lecithinase in the Presence of Polvmetaohosohate in
Acid Solutions:
A weighed quantity of the crude lecithinase yvas, taken up in several
drops of 0.5'N sodium hydroxide solution, and the slightly turbid solution
diluted with buffer to a protein concentration of 0.5 per cent.
Five
13/
meq. — ' of SPMP per gram of protein were added to half this solution.The remainder was used for controls.
Three ml. aliquots were taken from
10/ Specific activity refers to the MLD per mg. of nitrogen ratio in the
protein mixture.
ll/ A designation for the protein-polymetaphosphate complex. Perlman (17)
considers the reaction between PO3 and albumin.
12/ PMP, Sodium polymetaphosphate.
13/ I meq. of PMP equals 0.102 g. of the polymer.
both the SPMP solution and the control.
The pH of the aliquots was adjusted
with 0.5 N or 2 N sulfuric acid (the 0.5 N acid was added first and, if
necessary, the 2 N acid was used).
After the pH was adjusted the solutions
were diluted with water to a final volume of 4.5 ml.
One ml. portions of
the 4.5 ml. solutions were measured into tubes marked at 2 ml,, and the
tubes allowed to stand for the indicated acid contact times at room temp­
erature.
The precipitate was then dissolved in 0.5 N sodium hydroxide.
Results are presented in Figure I, 2 and 3,
2.
The Effect of pH on the Stability of Lecithinase-POg:
Crude lecithinase (0.5 per cent) plus one-half its weight of SPMP
was dissolved in the buffer at pH 8^9.
The protein alone in the buffer
gave a suspension, but on addition of the SPMP the liquid cleared.
Five
protein-PMP solutions were adjusted to the intended pH values (1-5), and
one ml. of each solution was placed in, a calibrated tube.
After the
indicated time interval, sodium hydroxide (0.5 N) was added to bring the
pH to 7-8, and sufficient isotonic borate buffer
the solution to volume.
was added to bring
The lecithinase activity was determined for 5 pH
values, two acid contact times, and two temperatures (3-5°C. and room temp,erature).
A second set of readings, was taken for the pH range 5-12.1 with
acid contact times, of 4 hours at room temperature.
The results are shown
in Figure 4.
14/ Rorate'buffer-, calcium chloride and sodium chloride solution at the same
cpncentrations as used in the lecithinase determination (see page' 6)..
-19—
3.
Lecitfainase Purification as a. Function of Acid Contact Time:
The lecitfainase precipitates were obtained as follows:
crude Ieci-
tfainase, specific activity about 75, plus one-half its weight of SPMP was
dissolved in 0.1 M )x 2-methyl-2 amino-1, 3 propanediol buffer.
was divided into three parts.
The solution
The pH of the solutions was then adjusted to
2.37, 3..72 or 5.0 with 0.5 N sulfuric acid.
were- then allowed to stand at 3-5°C.
Aliquots of the solutions
At a specified acid contact time,
one tube was centrifuged (RCF equals 1500), the supernatant discarded,
the precipitate washed twice with distilled water, and then dissolved with­
in 45 seconds by mixing with several dbops of 0.4 N sodium hydroxide.
Isotonic borate buffer at a pH of 7.5— 8.0 was immediately added and Iecithinase activity determined in the solution.
The nitrogen content of the
precipitate was determined by the method of Van Slyke (29).
by the method of Swingle (4).
Lecitfainase
The values ^ A i a v e been plotted in Figure 5.
4 . The p H at Which the Lecithinase-POo Precipitate Forms for one Ratio of
PMP and. Protein;
In the experiment in which an aqueous solution was used, bacterial
filtrate containing 1.0 per cent of unadjusted Calgon, (analyzing 75 per
cent polymer) and 24 per cent sodium chloride was adjusted to the desired
pH values with 0.5 N sulfuric acid and the solution allowed to stand at
-15°C. for three and one-half hours.
The tube was, then centrifuged,, the
precipitate separated from the supernatant, dissolved, taken up in borate
buffer and the lecitfainase activity determined by the method of Swingle (4).
15/ The- purification factor is the ratic of the specific activity of the
final material to that of the starting material.
-'20—
The supernatant solution was then used for the next separation at a lower
pH value. (See Figure 6).
5.
Purification of Lecithinase in Bacterial Filtrate at Acid pH Values;
Bacterial filtrate containing I 0O per cent PMPy 25 per cent sodium
chloridey and 12 per cent (v/v) glycerol was. swirled at -IS0C 0 during the
15 minute acid contact time.
For the first precipitation^ the pH was
adjusted with 1.0 N sulfuric acid; on subsequent precipitations of the
previous precipitate, 0.24 N acid was used.
The precipitate was dissolved
in borate buffer containing 25 per cent (v/v) glycerol and an aliquot of
the solution analyzed for lecithinase and nitrogen by the same methods as
were used in the aqueous -solution.
shown in Figure 6.
The data- for the aqueous experiment are
Figure 7 presents the data as obtained in the glycerol
experiment.
6.
Concentration of Components in the Crude Lecithinase-POn Precipitate as
a. Function of Concentration of Polvmetaohosohate and the pH of the
Solution;
Crude lecithinase,' about 15 MLD per mg. of protein, specific activity
of 94 (protein factor 6;25-),' was suspended in 2-methyl-2-amino-l, 3 propane-:
diol-acetic acid buffer (each at 0.1 M) at a pH near neutrality.
Then
several drops of 1.0 N sodium hydroxide were added to bring the pH to near
8,5.
(In this adjustment, care must be taken that higher pH values are
not used, since the lecithinase is more susceptible to inactivation at pH
values above 8.5).
The solution cleared to a faint turbidity.
Weighed
solid PMP (95.6 per cent polymer) was. then added and the solution was
rapidly swirled for several minutes for complete solution of the PMP.
The
-21-
solution became crystal clear.
Aliquots of the solution were then adjusted
to the desired pH with Chb N sulfuric acid which was added drop by drop with
swirling.
After an acid contact time of 10 hours at room temperature or
H O hours at 3^-5 °C.S the precipitate was recovered by centrifugation at
RCF equals IbOO and the supernatant liquid discarded.
The drained preci­
pitate was dissolved in Oib N sodium hydroxide, taken up in isotonic
buffer, pH 7.4, and the lecithinase activity, nitrogen content^ and phos­
phorus determined.
Lecithinase units are given in MLD- per mg. of nitrogen;
however, a primary standard was not available.
ratios are shpwn in Table I.
Results and calculated
Statistical data in the form of an analysis
of variance for F distribution significance are presented in Table II.,
The data have been plotted in Figures 8 to 14.
-22-
B4
RESULTS
The area between Curves I and 2, Figures I, 2% and 3 ? decreases from
an acid contact time of 4 hours to an acid contact time of 72 hours in the
pH range 2 to 4„
In general? Iecithinase recovery in a splution having a
pH below 4 decreased with an increase in acid contact time.
In each
figure it is seen that Curve I intersects Curve 2 between pH 4 and 5.
With increasing acid contact -time, the point of intersection approaches
pH 4„
At pH 5 recovery in the PMP solution becomes less, with increasing
acid contact time.
Lecithinase recovery in 2-methyl, 2-amino-L,- 3 propanediol-acetic
acid buffer as, plotted in Figure 4 shows the dependency of lecithinase
stability on the pH of the solution.
In the acid region-, curves for leci­
thinase recovery for different acid contact times and solution temperatures
were well separated.
Least lecithinase recovery occurred at pH 5-5.5 and
near 12 and at acid pH values at rpom temperature.
at pH, 7 to 10 and at acid pH values.
Good recovery occurred
Curve 3 with an acid contact time of
24 hours at 3-5°C . show's a lecithinase recovery of over IQO per cent.
Lecithinase purification as a function of acid contact time in Fig­
ure 5 shows the highest purification factor at pH 3.72, the lowest factor
odcurred in the most acid solution..
In Curves I and 2 , Figure 6, it is seen that nitrogen in the precipi­
tate attains a peak at one pH value and the maximum purification factor
shows a peak at a pH value about 0.5 pH units lower.
-
23-
Figure 7 shows a Teiatively large decrease in the nitrogen content
■of the precipitate as formed by reprecipitation of one precipitate.
The.
Iecithinase purification factor increased to precipitation number 3 after
which there was a decrease and then an increase in the factor.
The amount
of precipitate in the last precipitations made the determination of nitro­
gen ,difficult.
These values being somewhat in doubt; are indicated with a
-- dotted line on the graph.
The lecithinase in these precipitates contained
less than one per cent of the total starting material.'
In Figure 8 the specific
activity wa.s at a maximum for pH 4.5 and
2 meg. of PMP per g. of protein.
A slight-rise for1 the same polymer con­
centration also occurred at pH 3.5.
was obtained at 3-5°C.? Figure 9.
The best over all specific activity
Good recovery occurred over a wider
range of PMP (almost 0 to 2) at a pH value of 4-.5„
The MLD/mg. P curve at room temperature. Figure 10,■ showed a maxi­
mum at pH 4.5 and with 3 meq^ of PMP per g. of protein.
'?■
The ratio of MLD/
mg. P increased at a lower temperature, Figure 11, and the maximum was
obtained with a smaller amount of polymer (2 meq. of PMP per g. of protein
at pH 4;5).
Figures 9 and 11 show a rough.similarity in the general shape
of the curves.
The same observation can be made for Curves 8 and 10.
Figure 12 supports the general observation that size of the precipi­
tate increases with a decrease in pH of the solution.
In the intermediate
acid pH range, more nitrogen appeared in the precipitate at 3-5°C. than
at 250,C„
—24-.
In Figure IS5 the cross over between the curves' for the two temp­
eratures occurs at pH 3.5.
'
Figure 14 shows that the P/n ratio at room temperature varied with
the amount of polymer per g. of protein.
-25Table I
The concentration of lecithinase, nitrogen, and phosphorus in precipitates
obtained at three pH values and v'ari'ou s concentrations of
polymetaphosphate, (PMP) at room temperature
and at 3-5°C.
pH
PMP*
(meq./g.
orotein)
Lecithinase recovery
(MLD's/ootj (per cent)
N
'
(ma/Wt.)
MLD's
P
per
(mq/potJmq.N
Molar
MLD's ratic
per
of
mq.P P:N ■
Acid contact time 10 hours at room temperature
2.5
3.5
4.5
5 .0
2.5
4.0
440
440
325
■37
37
27
352
32
3 .5
415
4.5
345
37
31 .
2 .5
3 .0
3.5
4,5
2.5
3.5
4.5
2 .0
2 .5
1.0
3.5
4.5
2 .5
3 .5
0.10
4.5
2.5
0.01
3 .5
4.5
2.5
3 .5
4.5
340
290
30
38
25
435
0.00
345
32
380
35
310
28
700
1330
2710
0 .2 7
454
678
0.50
0.34
0.13
222
392
704
1220
704
2650
0.14
0.14
0.12
1.47
0.61
0.9 9
p .30
0 .4 2
0.09
196
438
557
1035
0.1 6
0.19
,9 6 7
3190
0.14
I..40
0.-71
p.21
0.66
0.47
0.16
246
536
523
0.-21
1510.
810
1940
0 .3 0
0 .3 4
6 .8 2
1 .2 0
0 .5 7
0.61
404
542
0 .3 3
0^48
0.17
297
740
1820
0.18
0.14
1340
1650
0.17
0.14
2540
0 .1 2
246
1350
O.QS
143
758
4500
0.14
4170
0.0 8
1.06
0 ,9 7
0.4 8
1.58
1.06
6 .4 9
0.63
0.3 3
0.12
330
34
355
310
36
32
335
365
330
32
35
31
0.66
0.25
0 .7 2
0.22
0.49
O.lS
54
'5
p .22
270
250
26
24
0 .2 0
0.04
0.06
0.-33
0.06
86
88
48
8
,8
5
0 .0 2
0.01
0.-01
415
548
506
505
676
5500
7000
3380
0.15
0.11
■;
* One meq. of sodium polymetaphosphate per gram of protein equals 0. 102
grams of polymer per gram of protein.
-26-
T able I continued
pH
PMP
(meq./g. Lecithinase recovery
protein) (MLD's/ppt.) (per cent)
M L D 's
N
P
per
(mq/ppt) (mq/ppt.) mq.N
Molar
MLD's ratio
per
of
mq.P
■P:N
Acid contact time H O hours at O0C.
2 .5
3.:5
10.00
4.5
2 .5
5.0
3.0
3 .5
4.0
4 .5
2.5
4;0
3 .0
3 .5
4.0
4.5
0.14
0.34
175
412
784
1120
1860
734
0.11
0.-16
0.19
0 .9 9
0.83
552
657
0.38
1.11
0.47
36
0 .2 8
0.06
484
1200
1260
1300
887
1310
1610
1950
0.2 5
0 .4 2
CU 36
8
0.61
0.43
0.2 2
0.04
545
655
62
1.61
1.68
0.55
0;82
0 .7 0
338
665
936
0 .2 3
0 .1 9
0.3 8
0 .3 0
0 .2 0
0 .03
69
74
67
42
8
575
64
-
0.01
0.46
391
1070
1240
1280
1850
-
2300
594
694
938
945
744
0.30
0.30
0.14
0,97
0.8 8
0.65
0.36
0.07
554
1.83
1.76
1.43
0.89
0,74
764
825
,1190
0 .6 4
0 .0 8
0.2 3
CU 02
371
348
411
904
2100
-
0.-15
0.16
0.11
1.76
2.04
1.57
0.9 7
0.46
329
274
598
0.2 5
584
0.56
350
65
36
0 .9 0
0 .32
0.09
642
982
1790
0.21
0.16
0.16
2000
3550
0.25
193
ki
0.79
878
0.13
44
0 ,6 8
0 .1 9
0 .2 2
0 .2 2
244
405
400
598
0.16
2100
1840
2500'
0 .1 4
0 .3 8
'610
610
340
3 .5
0.50
55
55
57
370
3 ,0
2.10
0.94
0.34
545
540
565
355
78
590
4.0
4.5
2.5
3.0
368
386
266
69
69
39
6
52
1.04
1.70
1.52
1 .08
0.4 6
338
402
315
113
0.40
0 .2 4
0 .1 9
0.15
0 .6 8
I
2 .5
3 .0
3 .5
2.0
610
585
580
4.0
4.5
2.5
680
168
1.0
580
3 .0
560
3.5
4.0
4.5
550
575
2.5
3.5
4 ;5’
0.10
86
'
•
77
74
;74
'21.
66
63
62
44
0.16
0.49
2520
0 .2 2
0 .1 9
-27Table
pH
I continued
PMP
(meq./g.
protein)
2 .5
3 .5
4 .5
0.01
2 .5
3 .0
0 .0 0
3.5
4.0
4.5
Lecithinase recovery
(M L D 's/ppt.) (per cent)
.Molar
MLD's MLD's ratio
per
of
per
N
P
Omq/ppt.)- (mq/ppt.) mq.N mq.P P:N
205
26
0 .5 0
590 .
600
75
0.49
0.42
14
2
3
12
25
28
0.21
28
103
210
238
76
0.-12
0.11
0.10
410
1200
1420
0.11
0.10
0.11
0.11
0.05
0.0010
1270
560
0 .1 0
0 .1 3
0.0010
0.0008
0.0010
0.0041
0.0054
0.0045
0.0006
1030
1610
1130
0.0028
0.0021
-28Table II
Analysis of variance of the data from Table I
Summary of F distribution, significance
Source of
Variation
Lecithinase
N
(MLO's/ootJ (mcV DDtJ
P
(mq/oDtJ
M L D 1S MLD's
per
per
mo. N ‘mq„ P
P:N
At room temperature
PH
Polymetaphospha te
*
**
, 0.1656
Linear regression
Quadratic regression
Control vs. rest
**
**
0,4396
**
**
-
**
**
0.4140
, **
*
0.7184 0,4152 ,0,5179
**
-
-
**
**
-
—
*
-X-X-
*
*
At S-S0C „
pH
Polymetaphosphate
**
0.5269
Linear regression
Quadratic regression
Control vs. rest
“
-
0.5847
-
*
* Significant at the 5 per cent level,
** Significant at the I per cent level.
Estimate of total error.
**
—
*
**
0.6638
**
*
*
1.0590 2.3670 0.5419
*
-
—
—
-
X
—
—
-
29
<0 sP
CURVE I
U W
CURVE 2
WITH PMP
CURVE I
W ITH O U T PMP
CURVE 2
PH
FIGURE I
ACID C O N T A C T T IM E 4 HOURS
CURVE 2
C UR VE I
FIGURE 2
PH
ACID C O N T A C T
T IM E 7 2
HOURS
CURVE
T
W
CURVE
UJ U
PH
FIGURE 3
ACID CONTACT TIM E 180 HOURS
FIGURES 3 , 4 AND 5
LECITH IN A SE RECOVERY( % ) IN SO LU TIO NS AT
A C lD p H VALUES W IT H AND W ITHOUT PMP
■CURVES
CURVE 2
CC 60
CURVE I
CURVE I
<
60
6 .0
7.5
SOLUTION pH
FIGURE 4
ENZYME RECOVERY IN Z-M ETHYLrZ-AM I NO
1, 3 PROPANEDIOL-ACETIC ACID BUFFER ( l) ACID CONTACT
TIM E 4 HOURS AT 2 5 cC (2 ) AC TIME 4 HOURS A T 3 - 5 e C
(3 )AC TIME 2 4 HOURS AT 3 - 5 °C (4 ) A C TIME 2 4 HOURS
AT 2 3 - 2 5 eC
16
££
R
pH 3 .7 2
5
z ao
o
pH 5.0
i-
<
U
§ 4 .0
a.
20
ACID
30
40
50
60
C O N T A C T TIM E (HOURS)
FIGURE 5 LECITHINASE PURIFICATION IN THE SODIUM
POLyMETAPHOSPHATE PRECIPITATE AS A FUNCTION OF
ACID CONTACT T IM E A T 3 - 5 °C
32
LU CU
CL OC
3 .0
FIGURE 6
CURVE
I
C UR VE
2
3.5
pH
LECITHINASE AND N ITR O G E N
RECOVERY IN P R E C IP IT A T E S
FIGURE 7 RECOVERY OF L E C IT H I NASE
BY REPEATED PR E C IPITA TIO N IN A
GLYCEROL SOLUTION AT pH 2.8
N
14
H
2.5
2.0
3.0
4 .0
POLYMER (M EQ ) PER G. OF PROTEIN
FIGURE 6
LEC ITH IN ASE A C TIVITY PER MG. OF
NITROGEN AS A FUNCTION OF C O N C EN TR A TIO N
OF SPMP AT A CO NSTA N T pH AND ROOM
TEMPERATURE
M L D 1S PER
MG. OF N IT R O G E N
34
.
0
I
1.0
POLYMER
i
i
2.0
(M E G )
i
i
3 .0
4 .0
PER G OF P R O T E IN
5.0
FIGURE 9
L E C IT H IN A S E A C T IV IT Y PER MG OF
N IT R O G E N AS A F U N C T IO N OF C O N C E N T R A T IO N
OF SPMP A T C O N S T A N T p H A N D 3 - 5 °C
5 0.5
POLYMER (MEO) PER G. OF PROTEIN
FIGURE IO
LECITHINASE ACTIVITY PER M G OF
PHOSPHOROUS AS A FUNCTION OF THE SPMP
CONCENTRATION A T A CONSTANT pH A N D
ROOM TEMPERATURE
(8 4 0 0 )
Y IO
<m
45 L
M L D 'S PER MG. PHOSPHORUS
40
35
30
25
20
OJ
0>
pH 4 .5
15
IO
5
1.0
2.0
3 JO
4 .0
POLYMER MECL PER G. OF PROTEIN
5.0
FIGURE I l LECITHI NASE ACTIVITY PER MG. OF
PHOSPHORUS AS A FUNCTION OF CONCENTRATION
OF SPMP AT CONSTANT p H AND 3 - 5 e C
CM
N
FIGURE 12 PROTEIN NITROGEN IN THE PRECIPITATE
AS A FU NC TIO N OF SOLUTION pH IN THE PRESENCE
OF 2 MEQ. OF POLY METAPHOSPHATE PER G. OF PROTEIN
1.0
.AT ROOM
✓
tem perature
3.5
SOLUTION pH
FIGURE 13 A M O UN T OF PHOSPHORUS IN THE
PRECIPITATE AS A FUNCTION OF pH IN THE
PRESENCE OF 2 M E O OF PMP PER G. PROTEIN
PRECIPITATE
0.4
P /N
RATIO IN
2 .5
04
3.5
4 .5
B A S E L IN E IN D IC A T IN G T H E P Z N R A T IO F O R PR O TE IN
A N A L Y Z IN G
3 eZe P H O SP H O R O U S A N D 16 e Z o N IT R O G E N
POLYMER
MEO
PER GRAM OF PROTEIN
FIGURE 14
P / N RATIO AS A FUNCTION OF SPMP
C O N C E N T R A T IO N AT CONSTANT pH A N D ROOM
TEMPERATURE
CO
-40'
C.
DISCUSSION
The previously presented data offer an estimate of the possibilities
and limitations for the acid precipitation method of purifying the Iecithinase in the presence of PMP.
I.
Protective Action of PMP on Lecithinaset
At four hours acid contact time?- Figures I, 2, and 3, PMP exerted
a protective action on the lecithinase in acid solution; that is, the
complex was more resistant to loss of biological activity and a precipi­
tate of higher specific activity can be obtained by including PMP in the
lecithinase solution.
With increasing acid contact time-,- bonding between
protein and PMP was probably decreased.
Depolymerization of a PMP solution
at room temperature and pH 2 was found to be less than one per cent after
10 hours of acid contact.
At 96 hours, 15.5 per cent of the PMP depoly-
merized (at 0°C. pH of I, 14.5 per cent of the PMP was lost after 120 hours).
It was also determined that crude lecithinase did not demonstrate any
phosphatase activity.
In this experiment only one concentration of PMP was investigated.
Since an equilibrium exists between the protein-POg complex and soluble
PMP9 increasing the amount of PMP in solution might extend the protects
ive action of the PMP.
Addition of acid to the lecithinase solution initially at pH 5, Fig­
ures I9 2, and 3 with or without PMP resulted in formation of a precipi­
tate in the pH range 4 to 5.
On this basis the isoelectric point of the
lecithinase could be- estimated at about'4,7,
According to,Briggs (14),
. -41-
the isoelectric point of the lecithinsse-BOg complex shifts to the acid
side with increased PMP content.
That the lecithinase-POg is probably formed
at pH values above this isoelectric point (4.7) is ,shown by the work of
Putnam and Neurath, (22) who found that sodium dodecysulfate formed two
complexes with albumin at pH 6.8.
The'low lecithinase recovery with PMP
at pH 5 indicates that in these solutions the lecithinase-POg complex is,
more susceptible to denaturation than is lecithinase alone.
2.
The Stability of Lecithina se-POg in 2-methvl-2-amino-1.3 propanediol
Buffer:
Recovery of more than 100 per cent of the original lecithinase
activity, Figure 4y when the enzyme in solution is allowed to stand at
low temperatures is a frequently encountered phenomenon.
This effect might
be explained by assuming that removal of extraneous protein from biological­
ly active centers on the lecithinase molecule occurs.
As would be expected, at high pH values the ,enzyme was least stable^
as the pH of the solution approached that of physiological pH (7.33), leci­
thinase stability was near maximum.
At pH 5.5 most of the protein was
still in a soluble form, and without the stabilizing effect of insolubility
inactivation occurred.
As the pH of the solution was lowered, more of the
lecithinase separated from the solution in insoluble form and became less,
susceptible to denaturation.
Increasing the acidity gave more rapid denat­
uration indicating that the stabilizing effect of the precipitated form no
longer balanced the denaturing action of the strong acid.
The 2-methyl-2~
amino-1,3 propanediol buffer, pK 8.78 (46), was included in the system to
prevent the solution pH from becoming to basic while adjusting the pH of
-42-
lecithinase solution or dissolving the lecithinase-POg precipitate,
3,
Lecithinase Purification as a Function of Acid Contact Time:
The nitrogen cpntent of the precipitate,Figure 5, between one arid
two hours of acid contact time increased six-fold, the lecithinase acti­
vity threesfold.
Comparison of the amount of nitrogen in the precipitates
formed from two to 72 hours showed a standard deviation of 0,044„
The
standard deviation for lecithinase content of the precipitate in per cent
of the original for the same acid contact time is 6.1,
Hence the rise
occurring between four and eight hours for the solutions at pH 3.72 is
probably the result of reactivation of the lecithinase.
Thus within certain
limits it could be expected that longer acid contact times would give a
higher specific activity.
4.
The Separation of Lecithinase Protein from Nonlecithinase Proteins
Figure 6 shows a good separation between lecithinase and total protein;
hence, the possibility exists for increasing the specific activity of leci­
thinase- POg by the method of acid precipitation.
In this series of investi­
gations,■ losses of lecithinase activity varied from about .15 to 36 per
cent.
_ JBelow pH 4
the left slope of the curve appears to be greater than
that on the right side of the peak.
This decrease in purification factor
may result from denaturation because of the relatively longer acid contact
time to which these solutions were subjected.
Putnam and Neurath (21) found less combination between bovine serum
albumin and dode.cylsulfate with more salt in the reaction mixture or with
reduced temperature.
Stabilizing action of PMP on lecithinase in the .
presence of 25 per cent salt' might then be less because of reduced com­
-43-
bination between protein and PMP.
Turoin et al. (7) repeatedly emphasize
the necessity for use of low solution temperatures (-15°C. or less.) in the
purification of the toxins and antitoxins.of certain bacteria.
However,
it would seem that the good recoveries which these authors obtained are
certainly not dependent pn complete saturation of ,all free amino groups
in their proteins with the protective §gent.
Preliminary tests on crude
Iecithinase-POg have shown little temperature inactivation to about 25°Cj
with an acid contact time of three and one-half hours.
5.
Purification of Lecithinase by Reprecipitation;
I t 1is seen that subjecting one precipitate,Figure 7 ? to a number
of subsequent precipitations at first increases the specific activity
and there then results.a decrease'In recovery of lecithinase.
This
method of increasing the specific activity, of the precipitate is pro­
bably limited by the increased possibility of denatpration, which results
from alternately dissolving and precipitating the enzyme.
of the solution might be involved to a greater extent.
Also agitation
Preliminary experi­
ments indicated that in agitated solutions of high acidity the presence
of PMP actually increased the relative loss of lecithinase activity.
Agitation with PMP at 3-5°C. ip the pH range 4-8 gave little or no loss of
lecithinase activity.
Agitation of the lecithinase with or without PMP at
room temperature resulted in loss of lecithinase activity.
6„
Influence of PMP Concentration on the Comoosition of the Acid
Precipitates
At room temperature, the amount of PMP present in the starting solu­
tion influenced .both the anoint of all listed components in the precipitate
-44-
and the calculated ratios.
However-, the ratio of MLD per mg. of phosphorus
showed significance at the fiye per cent leyel only.
F o r ■solutions at
3-5°C., the amount of PMP showed no significant relationship to the com­
position of the precipitate except for MLD per mg. of phosphorus, and the
P/N ratio (this at the five per cent leyel).
The lack of significance at
3-5°C. for the relationship of concentration of initial polymer in the
solution to phosphorus in the precipitate demonstrates that the point of
equilibrium between PMP and protein is probably temperature controlled,
Putnam and Neurath (21).
7.
Influence o£ ,pH on the Amounts of Various Components in the Precipitate;
.At both temperatures, the pH influenced the amount of nitrogen and
phosphorus in the precipitate (p % 0,01).
Lecithinase activity of the
precipitate was. significantly influenced by pH at room temperature (p % 0.-05)
-as
was the ratio of MLD per mg. of nitrogen at 3-5°C. (p
0.05).
All
other pH relationships showed no significance at the 5 per cent level,
A
statistical analysis was not obtained for the influence of temperature on
the amount of components in the precipitate.
8.
Interpretation of the data in.ilables I and %I as Plotted in Figures .8
to 14:
'
Because statistical evaluation showed that the amount of PMP in­
fluenced the amount of various, components in the precipitate in a highly
significant manner for the solutions at room temperature, PMP (in meq. per
g, of protein) was plotted against other components in Figures 8 to. 14 for
both temperature ranges investigated.
-45-
The influence of the amount of polymer on the MLD per mg. of nitro­
gen ratio at room temperature as shown in Figure 8 was apparently profound.
This is indicated by the high peak for the specific activity ratio at 2 meq.
of polymer per g. of protein for the solution at pH 4.5^
A smaller peak
occurred for the same polymer concentration for the solution at pH 3.5.
The smaller peak might indicate a lap over in which lecithinase was
linked by the PMP to material which was insoluble at this pH; if so^, sharp
separation of lecithinase from other proteins would be difficult by ,the
method of acid precipitation. ^At pH 2.5 most of the lecithinase and nonlecithinase protein appeared in the precipitate.
The slope of the curve
probably indicates denaturation of lecithinase.
The data plotted in Figure 9 enable some evaluation of the same
relationships at 3-5°C.
fluctuations.
nitrogen ratio:
These data show a less regular curve, with wide
.Twg,"'"factors are probably influencing the MLD per mg. of
first, the possible unmasking of enzymatically active
centers; and-, secondly, the probable reduced combination of PMP with
protein at lower temperatures.
9.
The MLD per mg,.
Phosphorus ratio as Influenced by the Amount of PMPs
In Figures 10 and 11 it is seen that in general the data for MLD
per mg. of phosphorus vs. the amount of polymer in some respects seem to
parallel that for MLD per mg. of nitrogen vs. the amount of polymer with
respect to shape of curve.
This might be expected,, because the linkage
in the protein-PO^ complex is between free protein amino groups in cation
form and the negatively charged POg monomer, a Isl ratio..
-46'
10.
Nitrogen and Phosphorus in the Precipitate as a. Function of Solution
pH: Figure 12 shows that protein nitrogen once precipitated at a higher
pH value remains insoluble as the acidity of the solution increases.
To.
the left of the cross over pH where the two temperature curves intersect^
more protein nitrogen appeared in the precipitate at 3-6°C. than in the
solution at 25 0C..
For I meq. to 3 meg. of PMP per gTi of protein9 the
pH of the cross over point increased in a nonlinear manner.
The solution
pH values were initially adjusted at ropm temperature; hence, the actual
acidity of the solutions at 3-5°C. would differ somewhat from the values
shown in the figure.
At these cross over pH points,* the nitrogen content .
of the precipitate was independent of the temperature.
In Figure 13, it
is seen that the phosphorus content of the precipitate was independent
of the temperature at pH 3.5.
This may mean that the most stable link­
age between protein and PMP occurs at these cross over pH points.
The next question is whether these cross over pH values correspond
to the probable .isoelectric point of the lecithinaSe-PO3 complex; that
is, the pH at which the lecithinase complex should be most stable..
To
adjust the isoelectric point of the lecithinase from about pH 4.7 to a
more acid pH value should require progressively more PMP per g. of pro­
tein.
The cross over pH point"for I to 5 meq. of PMP per g. of protein
(5 graphs) varied from pH 3.5 to pH 4.4 and did not show a regular change
in cross over pH with increase in PMP concentration.
Two meq. of PMP per g. of protein does, however, seem to be a
significant value.
In Figure 14, the P/N ratio at room temperature at
-47-
P'H 3.5 and 4.5 attained a maximum at 2 meq. of PMP per g„ of protein.
In
Figure 8 and 9 , the specific activity of the lecithinase was at or near
maximum for 2 meq. of PMP per g. of protein.
It is apparent that in
certain ranges of PMP per g. of protein, a small change in PMP concentra­
tion results in a considerable change in thej^ lecithinase content of the
precipitate.
Saturation of the lecithinase with PMP in the sense that all
available reacting sites on the protein are combined with PMP would diff•
{
er from saturation of the protein with PMP in the sense that under cer­
tain conditions.an equilibrium between complex and protein plus PMP
favored the formation of free protein and PMP.
At room temperature with 5 meq. of PMP per g. of protein, only a
fraction of the polymer added (one-fourth to more than one-.half according
to pH) appeared in the precipitate.
With 2 meq. of polymer per g. of
protein, the precipitate contained more phosphorus, natural and added,
than was initially present in the PMP.
Phosphorus values in the precipitate on the controls at room temp­
erature were not reported because of experimental difficulties.
-48-
D.
CONCLUSIONS
A lecithinase precipitate of high specific activity can be obtained
by using a ratio of 2 meg. of PMP per g. of protein in 2-methyl-2-amino-l?
3 propanediol-acetic acid buffer and an acid precipitating pH of 4.5 at a
temperature of 3-5°C.
A short or long acid contact time with a minimum
of agitation could be used.
With a long acid contact time, reactivation
of the lecithinase occurs.
Starting with the bacterial filtrate, the first precipitation of
lecithinase with ammonium sulfate can be expected to increase the specific
activity after dialysis and lyophilization of the precipitate by a factor
of about 10.
The overall purification factor would be 220 fold.
This
value is in accord with that observed in' the data obtained by acid
precipitation of lecithinase in the presence of PMP from the bacterial
filtrate.
In these solutions purification factors between 27 and 221,
depending on conditions were obtained.
It is. evident that acid precipitation of lecithinase in the presence
of PMP offers possibilities as a:means of obtaining lecithinase protein
of high specific activity.
t
PART H s
THE SEPARATION OF CRUDE LECITHINASR ON THE DIETHYLAMINOETHYL
CELLULOSE COLUMN
Preliminary determinations by the Lowry method (Si) for protein in
TRIS buffer have shown that the protein value is influenced by the amount
of TRIS buffer in the reaction mixture; however, the TRIS buffer absorbed
only slightly at the wave lengths, used in the Warburg method (32) of
determining protein.
Separation of the buffer from the protein by dialysis
would result in about a 22 per cent loss of lecithinase activity in the
column eluates.
This lecithinase loss can be tolerated if the activity
of the protein placed on the column is of the order of that used, in this
experiment.
It was further determined that saturation of DEAE with crude
lecithinase protein will not occur belpw a ratio of about 9 mg. of pro­
tein per g. of DEAE.
A-.
1.
EXPERIMENTAL PROCEDURE
The Column Material:
The column, material was Eastman DEAE cellulose, catalog number 7392,
which was purified according to the procedure of Moore and Lee (45).
purified D.EAE analyzed approximately I,.3 per cent nitrogen..
The
The DEAE
(3.2 g.) was suspended in 0.005 M TRIS buffer at an initial pH of 7.75.
The slurry was then poured into the 1x20 cm. glass tube.
After each
increment of slurry, the slowly forming column was packed under a pres­
sure of about 5 psi with pompre.ssed air.
2.
Eluent Buffers:
The composition of the eluent buffers- is shown in Table III.
-50,Table III
The composition of eluent buffers for the DEAE column.
Number of tubes
of Eluate
Exoected
Actual
Buffer
Number
I
5
10
.10
10
10
15
2
3
4**
5*
*
6**
6
10
10
10
10
15
TRIS*
(Molaritv)
Sodium Chloride
(Mblaritv)
0.005
none
CLOl
0 .0 5
0 .0 2
0.10
0.05
0.05
0.05
0 .1 5
0;25
0 .50
Final
Volume
(ml;)
• .50
100
100
100
100
150
dH
7.80
7 .5 0
7 .2 0
7 .0 0
7 .0 0
7 .0 0
600
*
The Sigma 7-9 product was twice crystalized from water
Buffers 4, 5, and 6 were made 0.02 M with respect to calcium I
chloride
to stabilize, as much as possible, lecithinase remaining in contact.
with DEAE cellulose for longer periods of time.
Before elution, protein from any bacterial growth in the buffers, was
determined by ultraviolet absorption'at 260 and 280 nyj.
cejDt.■ number 4 showed negligible absorption.
All buffers ex-
Absorption of buffer number
4 showed a protein equivalent of 28 pg. per ml;
Gitlin (50) reports the
sensitivity of the UV method of protein determination as being 90 pg. per
ml.
3.
Hence, this value of 28 yg. was initially taken as being insignificarit.
Protein Placed on the Column;
The history of the crude lecithinase which was placed on the column
is given in the Preparation of Materials section, pages 10-14, and in
Table IV.
In Table IV lecithinase units as determined in column eluates refer
to enzyme activity but are not to be taken as being synonymous with MLD„
The lecithinase activity in the solutions which are mentioned in Table IV
-51-
was determined by antitoxin precipitation.
A portion of the Seitz filtrate,
mentioned in Table IV5 was diluted with glycerol until the final glycerol
solution contained 100 such units per ml.
This glycerol standard was then
used to determine the sensitivity of the egg yolk substrates which were used
to evaluate lecithinase activity in the DEAE column eluates,.
Table IV
The purification of the crude lecithinase previous
to placing it on the DEAE column.
Lecithinase
Fraction
Biuret
Protein
mq./ml.
Lecithinase*
Units/ml.
Lecithinase
Units/mq. N
Seitz
Filtrate
14.5
220
Dialysee**
12; 8
1995^2090
160.
1.9
8407-.8550
4450 .
Supernat-****
ant
*
**
***
, x*xx
Purification Factor
(over orevious step)
15.2
,10.5***
27.8
By'antitoxin precipitation.
After dialyzing out ammonium sulfate.
OveraI!-includes gain by ammonium sulfate precipitation and loss due
to dialysis. Estimated gain for ammonium sulfate precipitation,
about 18 times.
From nucleic acid precipitation; referred t° as Sol. 6.
It is seen that this protein sample has already been purified 292 times.
The crude lecithinase protein Sol. 6 (4.05 ml.), which contained a
total of 7.61 mg. of crude lecithinase (according to the Biuret method)
and 36,138 lecithinase units, was made 0.005 M to TRIS buffer and the pH
of the solution adjusted to 7.75 with hydrochloric acid.
The protein
solution was placed on the column in the cold room (3-5l0C.) by adding a
portion of the solution to the free space above the DEAE and then forcing
-52-
the solution do,wn with a hand bulb.
When the solution level was just above
the ,DEAE, several portions of 0.005 M TRIS buffer at pH 7.75 were used to
wash the protein into the c.olumn material.
A control sample of the pro­
tein was saved and this lecithinase subjected to the same temperature
conditions as were encountered by the column lecithinase.
4.
Connecting Tubing;
Tests showed that gum rubber connecting tubing would release com­
pounds which would develop a color with Lowry's (31) reagent; hence,
polyethylene tubing was used in making glass-to-glass connections.
5.
Mixing Chamber;
A. Mariotte mixing tube (24) of about 6 ml. volume was connected
between the buffer reservoir and the DEAE column.
This chamber was used
to effect a smoother gradient elution between changes of buffer in the
step-wise procedure.
Figure 16 shows that the increase in ionic strength
of the buffer was nearly linear to 150 ml. of column eluent..
With addition
of the calcium chloride solution the slope of the curve increased, but a
nearly straight line was obtained from 155 ml. to 350 ml. of eluent.
6.
Column and Dialysis Operation;
Column fractions were collected in volumes of about 10 ml. each.
Pressure (l-2 pounds per square' inch) was used on the. column.
The frac­
tion collector in the cold room was set to change tubes every eight
minutes.
A total of 62 samples were collected over a time interval of
about eight and one-half hours.
From each fraction a 4 ml. sample was
measured into a cellophane sack which, was then string tied.
The eluate
-53-
samples were dialyzed at 3-5°C. with stirring against 0.02 M calcium chloride
solution for approximately 24 hours.
The first 35 eluates were dialyzed
against 6 liter portions of salt solution which was changed three times.
The next 27 eluates were dialyzed against a comparable volume of calcium
chloride in portions.
could be obtained.
The undialyzed eluate was frozen until the pH values
The pH of the dialyzed samples was about 5.6,
completion of dialysis, the samples were frozen at -15 to -IT0C.
After
They
were then thawed and analyzed for lecithinase activity and protein content
at a later time.
The protein content of the eluates was determined by the
Warburg (32) and Lowry (31) procedures.
After the eluate fractions were collected, the DEAE cellulose in the
column was removed and teated with 10 ml. of I N sodium hydroxide, the
resin removed by centrifugation, and the ultraviolet absorption of the
solution measured at 260 and 28,0 mp.
-54B.
RESULTS
Values obtained in analyzing the eluate fractions are given in Table
V.
The Warburg protein values for column eluates were not included because
a graph of le.cithinase activity vs. eluate tube number for the Warburg data
seemed to show the presence of some interfering material? perhaps nucleic
acids.
Because of this interference, peaks on the graph were rounded
and troughs filled in.
Graphs of pH, lecithinase activity, and protein are shown in Figures
15,- 17, and 18.
The graph of lecithinase activity vs. eluate tube number
shows lecithinase activity in tubes 1-38 only.
No lecithinase activity was
found in tubes 39 to 62, and the graph was not extended.
The control con­
tained 30,678^7 units per 4.05 ml. at the time of stopping the fraction
Collector.
At completion of lecithinase analysis, the control contained
21,991.5 units per 4.05 ml.
Calculation revealed that 18,6 per cent of the starting lecithinase
activity remained in the dialyzed eluate fractions.
Meanwhile the control
in just standing at comparable temperatures lost, 39:2 per cent of its leci­
thinase activity.
It was estimated from dialysis experiments that the leci­
thinase should have lost 23 per cent of its activity.
The loss of lecithi­
nase activity is then 19;2 per cent more than expected.
When the DEAE cellulose was treated with I N sodium hydroxide, 1:2
mg. of protein were recovered as. indicated by UV absorption.
In the Lovvry
method, about 41 per cent,of the starting protein was accounted for.
Since
the column at addition of the sodium hydroxide was still moist with TRIS
puffer, a Lowry protein, determination was not run.
-55Table V
Analysis of column eluates for lecithinase and protein
Fraction
Number
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Fraction
Volume .(ml,)
10,0
8 ;0
9 .0
8 .3
8.7
9 .D
8.-4
10.0
9 .8
9 .8
9 .8
10.0
'
9 .0
22
23
9.5
24
9.6
25
26
9 .0
7 .5
8.0
■ 9:7
9 .9
27
28
29
7 .80
8 .0 0
8.25
8 .4 7
8.5
7.4
7..'5>
12.5
12.0
13.0
12.5
12.0
9 .4
Lecithinase
(units per
fraction)
Fraction
-d H
.46
3%
278
32
33
34
10.2
9.0
I O'.4
, 7.40
35
36
9 .8
9 .8
7.29
7.25
37
38
39
9.5
10.4
9.7
. 7.25
40
41
10 .0
9 .7
7.25
7.23
39
24
24
20
'
7 .5 2
7.5 0
7.48
7.68
7.59
7 .4 0
53
35
39
8.15
7.50
83
165
93
145
54
9.10
9.10
9.-10
9.10
9 .4
107
25
0
0
6 .5
9.1 5
9 .2 0
9.1 3
10.4
225
200
95
7.80
9.00
30
31
.43
878
3900
680
7.93
7.6 2
9 .2 0
9.19
9.2 3
9.35
9 .4 0
9.3 7
9.2 8
9.2 8
9.19
0
.1
Ool
8.00
'
Protein
Lowry*
(u q ./fraction)
17
18.9
32
16.4
1.6
68
39
88
17
10
36
25
-
'
71
22
14
16
1.5
- 11.3
16
2.4
15
2 .8
.93
1.4
-.56
2.1
1.2
1.6
1.8
15
45
83
7 ,6
4 .3
2 .4
1 ,3
41
31
1.7
31
0
0
2.7
7,24 '
4 .9
7.2 9
1.3
.
5 .2
.62
.90
8.1
12.5
.40
4 2 .2
-56Table V continued
Fraction
Number
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
Summation
values
Fraction
Volume (ml.)
'10.2
10.3
10.0 .
9,3
'9.7
10.2
10.8
10.5
11.4
10.4
10,5
9.4
10.8
9.8
10,,3
10.0
10.8
10.0
10.5
9.0
6.5
Fraction
dH
7.23
7.21
7.19
7.15
7.15
7.13
7.13
7.13 '
7.13
7,15 '
7.40
7.40
7.40
7.29
7.22
7.20
7.19
7.18
7.18
7..15
7.16
Lecithinase
(units per
fraction)
.22
.86
1.0
IiO
'
1.0
.42
.22
.44
0
1.3
.48
0.10
0.24
0.21
0.11
0.10
0.23
0.10
0.22
0.20
0.27
Protein
Lowry^
(ucu /fraction)
8.5
5.2
0
7.9
0
1.4
1.8
13.1
72
31
32
20
9.1
19.6 '
15
23
1.8
1.3
0
4.5
.65
607.3
6,737.6
1,907
* The Lowry standard curve for protein (absorbancy vs. ug. ) was obtained
by first plotting absorbancy vs. ml. of Sol. 6. Then a final curve was
plotted in which ml. of protein were converted to ug. of protein by
using the 1.88 mg. protein/ml. ratio.
a. 8.0
>0
24
COLUM N
FIGURE
15
28
32
FRACTION
36
40
44
NUM BER
HYDROGEN ION CONCENTRATION AS pH
IN THE ELUATE FRACTION
48
52
56
60
58
STRENG TH
BUFFER 5
IO N IC
BUFFER 4
WITH 0.02 M
BUFFER
BUFFER 2
ELUATE VOLUME (M L'S)
FIGURE 16
IONIC
STR EN G TH OF C O LU M N
ELUATES
BUFFER I I BUFFER 2
BUFFER 3 | BUFFER 4 | BUFFER 5
L E C IT H IN A SE
UNITS
|
IO
14
18
22
26
30
34
38
C O LU M N FRACTION N UM BER
FIGURE 17
A N A LY SIS OF CO LUM N ELUATES
FOR LECI TH I NASE A C T IV IT Y
42
46
225.
i=
175.
u-
150
.
125
z ioo
12
16
20
COLUM N
FIGURE 18
24
28
FRACTION
32
36
NUMBER
LOWRY PROTEIN CONCENTRATIO N IN COLUMN
ELUATES
-61C.
DISCUSSION
The discrepancy between amount of protein placed on the column and
the amount of protein determined in the dialyzed eluate by the two methods
of analysis was great.
Since there is no accurate method of determining protein concentration
in a mixture such as is encountered in the crude lecithinase, it was
thought that analysis of the eluate by the ultraviolet procedure of War­
burg (32) and by the Lowry method (31) would enable a more accurate inter­
pretation of protein content than one based on a single method of protein
determination.
With regard to the ultraviolet method of determining pro­
tein content of a solution, it has been observed in previous work that
protein concentration curves (absorbancy vs. mg. of protein solution)
obtained on dialyzed crude lecithinase usually failed to intersect the
zero point on the curve.
tion.
This fact is an indication of protein denatura-
Glazer and Smith (51) have determined the effect of protein de-
naturation oh ultraviolet absorption of protein before and after de­
nature tion.
They found that "In addition to difference peaks due to
tyrosine and tryptophane side chains at 278 mp. and 285 my. and 293 mp.,
respectively, denaturation was invariably associated with the appearance
of a far more prominent peak.at 230 to 235 mp.,K
As quoted by Nielands and Stumpf (52), a ratio of absorbancy 280/
absorbancy 260 of 1.6 or larger indicates the absence of appreciable amounts
of nucleic acids (nucleoproteins).
Values for this ratio on column eluates.
were frequently less than one; hence, the concentration of protein as read
from the Warburg monograph would be in error with excess nucleic acids.
62-
It would be expected that denaturation of the protein or the presence
of nucleic acids would change the protein value of a sample as determined
by the two methods.
That the extent of the change should be the same for
both methods is not probable.
It is apparent that peaks of lecithinape activity occurred in frac­
tions number 4, 9*. 20, and 3.1.
From the protein values obtained by the
Lowry method on column eluates, protein peaks occurred in factions
number 3, 10, 20, 31, 41, and 50.
A rough estimate of the areas under the
respective curves indicates a purification factor of 2-3 for the first
three lecithinase peaks.
The results suggest that there are three separate lecithinases.
The
plot of lecithinase units versus column fraction number shows three defi­
nite peaks and a possible fourth.
It is. noticed that the height of these
peaks decreases as the fraction number increases.
It is possible that
a portion of the lecithinase which should have appeared in one peak was
displaced for some reason to another peek.
However, the fact that leci­
thinase, concentration in intermediate tubes approaches zero would seem
to nullify this assumption.
The decrease in, peak height could result
from inactivation of lecithinase with increased DEAE contact time*, or the
second and third lecithinases could be present in the starting material in
lesser amounts, or their activity could be less.
Figure 17 shows that
these peaks of lecithinase activity occur about two or three tubes after a
change of buffer.
The column was operated under a pressure a little above
atmospheric pressure.
During a change of eluent, the compressor was dis-
63-
qonnected from the column and the column was then at atmospheric pressure
for a time interval of about 45 seconds.
Cassidy (53) states that column
elution once started cannot be interrupted without changing the shape of
the profile curve.
It is possible, then, that each change of eluent solu­
tion resulted, in more enzyme of the same species appearing in the eluate.
The complete lack of trailing between Reak I and Peak 2 (Figure 17) sup­
ports- the contention that two different lecithinases are present.
Trail­
ing was encountered between Peaks 2 and 3; hence, the same enzyme might be
involved in tube 9 and tube 20.
There was no trailing between Peaks 3 and
4.
An approximate calculation of the ionic strength of the six buffered
solutions which were used to elute lecithinase from the DEAE column shows,
as plotted in Figure 16, a nearly linear relationship between the ionic
strength of Buffers I, 2, 3.
To insure that lecithinase would be removed
from the column as rapidly as possible in the second half of the elution,
the ionic strength, of Buffers 4, 5, and 6 was increased above this, linear
relationship.
These buffers contain calcium ion (.02 M) to redupe inacti­
vation of lecithinase on the DEAE.
The calculated activity of the starting protein was 36j 138 units
(Vol. of solution X Units/ml.).
A recovery of 6,737 lecithinase units
indicates a loss of 81.4 per cent.
It is felt that this, lass is excessive
and that better recoveries should be possible.
-64-
D.
CONCLUSIONS
The rate of migration of crude lecithinase on the DEAE cellulose
column differs for- several lecithinase fractions.
The assumption is made
that this indicates the presence of two or more different lecithinases which
are
.elaborated
by the Clostridium hemolvticum bacteria.
It seems
probable that the best application of the DEAE column with crude lecithinase
will be to separate these lecithinase fractions.
GENERAL CONCLUSIONS
The separation of crude lecithinase protein on the DEAE cellulose
column does not give a large purification factor.
A comparison of the area
occupied by the curve as obtained by plotting lecithinase activity against‘
tube fraction number and the curve obtained by plotting total protein
against tube fraction number, as. shown in Figures 17 and 18, typically shows
about a three fold increase in the purity of the lecithinase.
Acid precipi­
tation of the lecithinase in the presence of PMP will give an increase in
specific activity of about 220 fold.
A second disadvantage involved in the
use of the DEAE cellulose column is. the amount of eluent required to make
the separation; hence, the lepithinase is diluted, and removal of the excess
liquid is time consuming and will probably result in the denaturation pf
some of the lecithinase.
It is suggested, that a quick procedure for con­
centrating the lecithinase in the eluate from the DEAE cellulose column
would be to use the procedure of acid precipitation of the lecithinase in
the presence of PMP.
This procedure would probably work best with eluate
fractions which do not have a high salt concentration, and would probably
increase the specific activity of the lecithinase.
-65-
Further work should be done in characterizing the several lecithinases.
as separated on the DEAE cellulose column.
A comparison should be made be­
tween these lecithinases- to determine if there is a difference in lethal
or lecithinolytic9 activity and the ability to lyse red blood cells.
It
would also be interesting to find put if these eluate lecithinases differ
in the ability to cause an antibody response in animals? or im wave
pattern in electrophoresis, or ultracentrifugation.
Most especially it would be of value to know how many lecithinases
are elaborated by Clostridium hemolvticum.
The author is currently investi­
gating the relatively new method of Immunoelectrophoresis in which electro­
phoresis is used to separate the various proteins in an agar covered
slide.
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2
NS 78
M247
cop.2
McRoberts, D. E
The partial purification of
the lecithinase of Clostridium
Hemolvticum._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
WAMK
AHO Aoommmm
■i? & Jf ^
■it-
NOV 17 #
M S I S
Al SL41
dop. 2